This application is a competitive renewal of a Program Project Grant entitled, Intracellular pathogens and innate immunity, originally funded in 2004 and renewed in 2009 with two years of funding from the ARRA. The central problem under investigation is how intracellular pathogens are recognized by the host innate immune system and conversely, how pathogens avoid and/or manipulate the host response to promote their pathogenesis. The program consists of three projects and two cores. In Project 1, Portnoy proposes to extend his discovery that L. monocytogenes activates a host cytosolic surveillance pathway (CSP) leading to the expression of IFN- and co-regulated genes. During the past funding period, the bacterial ligand was identified as c-di-AMP, a newly discovered small bacterial signaling molecule that appears to be essential for bacterial growth, and is secreted through a bacterial multidrug efflux pump. The role of c-di-AMP during infection and immunity will be explored. In Project 2, Cox found that M. tuberculosis requires the ESX-1 auxiliary secretion system to activate the CSP, and he proposes to identify and characterize the bacterial ligand(s) and host factors that contribute to the response. In Project 3, Vance collaborates with the other two groups and focuses on the identification and characterization of host factors that control the CSP. He has already established a program of ENU mutagenesis and identified that the host protein Sting is necessary for the response to L. monocytogenes, M. tuberculosis, c-di-AMP, c-di-GMP, and DNA. Furthermore, in preliminary data, Sting appears to be the cyclic-di-nucleotide receptor. Core B, managed by Barton, is a mouse and ENU core that will continue to generate and breed approximately 25 strains of mice with single, double and triple mutations in relevant innate immune pathways. The core will also manage the ENU forward genetic screen and dispense potential mutant macrophages to the other project leaders to screen for host defects in innate immune pathways. The purpose of Core A is to ensure scientific progress and promote synergy by providing scientific, organizational, and administrative leadership, which will be accomplished by extensive review of scientific progress during monthly meetings of all the lab groups.